Hollow nanoparticle of NBsAg large protein for drug delivery

ABSTRACT

The invention provides a therapeutic drug that uses hollow protein nanoparticles displaying an antibody against a specific cell or specific tissue. The effectiveness of the drug has been proved by animal testing. The invention also provides a therapeutic method using such a drug. In a drug according to the present invention, a substance to be transferred into a cell for treating a disease (for example, a cancer treating gene such as a thymidine kinase gene derived from simple herpes virus) is encapsulated in hollow nanoparticles of a particle-forming protein (for example, hepatitis B virus surface-antigen protein that has been modified to lack its infectivity to hepatocytes and display an antibody). The particle surface of the drug displays an antibody, such as a cancer specific antibody, that recognizes an antigen molecule displayed on the surface of a specific cancer cell.

TECHNICAL FIELD

The present invention relates to therapeutic drugs usingantibody-displaying hollow protein nanoparticles, and to hollow proteinnanoparticles. The invention particularly relates to a drug whoseparticle surface displays bio-recognizing molecules such as an antibodyagainst a specific cell or tissue, and which contains particlesencapsulating a substance to be transferred into a cell for treating adisease, wherein the drug allows the disease-treating substance to bespecifically incorporated into a specific cell or tissue. The inventionalso relates to particles suitable for the drug.

BACKGROUND ART

In the field of medicine, there has been active research on drugs thatdirectly and effectively act on the affected area without causingserious side effects. One area of active research is a method known as adrug delivery system (DDS), in which active ingredients of drugs orother substances are specifically delivered to a target cell or tissue,where they can exhibit their effects.

Another area of active research is a technique of gene transfer to aspecific cell, which is now essential in the field of molecular cellbiology. With the genetic background of various diseases being revealedby the Human Genome Project, a method of highly specific gene transferto a specific cell or tissue holds great promise because, once themethod is established, it is applicable to the field of gene therapy.

In one known example of a gene transfer method to cells, uptake of genestakes place in the form of a giant molecule by endocytosis (calciumphosphate method, lipofectamin method). In another method, genes aretransferred through cell membrane pores that are formed by thestimulation of the cell membrane with an electrical pulse(electroporation method, gene gun method). Both of these methods arecommonly used in molecular biology experiments.

Despite the simplicity of these methods, they cannot be readily appliedto cells or tissues of internal body, because the methods involve directphysical contact with the cells and surgically expose the site of genetransfer. It is also difficult to achieve near 100% uptake.

A transfer method that is safe to use is a liposome method. The liposomemethod does not damage the cell and is applicable to cells or tissues ofinternal body. A problem of the method, however, is that the liposome,which is a simple lipid, cannot have a high level of specificity to thecells or tissues, and uptake of genes in vivo is far below the requiredlevel.

In a recently developed technique, a therapeutic gene is inserted inviral DNA, and the gene is transferred by an infectious virus. Themethod is innovative in the sense that it does not expose the site oftransfer, is applicable to individuals, and provides near 100% uptake.However, the method suffers from a serious drawback in that the virusnon-specifically infects a wide range of cells, transferring the gene tocells other than the target cell. Further, the method has a potentialrisk of unexpected side effect if the viral genome is incorporated inthe chromosomes. In fact, the method is not used in initial stages ofdisease treatment. Only the terminal patients can receive the benefit ofthe method.

In sum, none of the conventional gene transfer methods is sufficient tospecifically transfer genes to a target cell and express the proteintherein to produce a drug. To this date, there has been no effectivemethod of directly delivering a protein as a drug into a target cell ortissue.

Under these circumstances, the inventors of the present invention havepreviously proposed a method of specifically and safely delivering andtransferring various substances (including genes, proteins, compounds)into a target cell or tissue, using hollow nanoparticles of a proteinthat has the ability to form particles and has incorporated abio-recognizing molecule, as disclosed in International Publication withInternational Publication No. WO01/64930 (published on Sep. 7, 2001)(hereinafter referred to as “International Publication WO01/64930”), andin Japanese Publication for Unexamined Publication No. 316298/2001(published on Nov. 13, 2001). However, these publications do not fullydiscuss how the method can be used to develop drugs for the treatment ofdiseased cells or tissues (cancer, for example). Specifically, thedevelopment of drugs displaying a specific antibody for specific cancercells or tissues remains to be one of the most important goals to beachieved, particularly in view of the following problems.

Owning to the difficulty in specifically and safely delivering andtransferring a protein (drug) into a target cell or tissue, a greatburden has been put on the patients receiving treatment using such aprotein drug.

For example, for the treatment of viral hepatitis (hepatitis C inparticular), an interferon, which is one form of a protein drug, isadministered systemically through intravenous injection over an extendedtime period. Though the effectiveness of the treatment is wellrecognized, it has many side effects due to the non-specific action ofthe interferon, including high fever, loss of hair, tiredness, andimmune response, which occur every time the drug is administered.

The hepatocyte growth factor is known to be effective for the treatmentof liver cirrhosis. However, since systemic administration of the drugthrough intravenous injection may cause unexpected side effects, thehepatocyte growth factor is directly administered to the liver with acatheter. The use of catheter requires surgery, which puts a burden onthe patient if he or she must receive prolonged treatment.

The present invention was made in view of the foregoing problems, and anobject of the invention is to provide a therapeutic drug, proved to beeffective by animal testing, that specifically acts on a target cell ortissue with its hollow protein nanoparticles displaying bio-recognizingmolecules such as an antibody. The invention also provides a therapeuticmethod, and hollow nanoparticles for use in such a therapeutic drug andtherapeutic method.

DISCLOSURE OF INVENTION

The inventors of the present invention accomplished the presentinvention by successfully preparing different types of hollow proteinnanoparticles displaying an antibody, and by finding that hollownanoparticles displaying an antibody specific to the human squamouscarcinoma cell was effective in the treatment of transplanted cancerwhen a drug encapsulating a cancer treating gene in the hollownanoparticles was administered in laboratory animals through intravenousinjection.

That is, the present invention discloses a drug in which a substance tobe transferred into a cell for treating a disease is encapsulated inhollow nanoparticles of a protein-forming protein displaying an antibodyagainst a specific cell or specific tissue.

An example of such a protein is a hepatitis B virus surface-antigenprotein that has been modified to lose its infectivity to thehepatocytes and display an antibody. In eukaryotic cells, the protein isexpressed as a membrane protein on the endoplasmic reticulum andaccumulates thereon before it is released as particles into the lumen.With the antibody displayed on the particle surface, the hollownanoparticles can act as a carrier, delivering the substanceencapsulated in the particles specifically to a specific cell orspecific tissue. As used herein, “specific cell or specific tissue”refers to cells into which the substance encapsulated in the particlesis introduced by the binding of the antibody with an antigen displayedon the cell surface, or tissues as a collection of such cells into whichthe substance is introduced.

The pre-S regions (pre-S1, pre-S2) of the hepatitis B virussurface-antigen protein have important roles in the binding of HBV tothe hepatocytes. Thus, the hepatitis B virus surface-antigen protein canbe modified to lose its infectivity to the hepatocytes by deleting someof the amino acids in the pre-S regions. In this way, the substance inthe particles can also be introduced into cells or organs other than theliver.

When some of the amino acids in the pre-S region are deleted to removethe infectivity of the protein to the hepatocytes, the level ofexpression of the modified hepatitis B virus surface-antigen protein inthe eukaryotic cell varies depending on the deleted area of pre-Sregion. The level of protein expression in the eukaryotic cell tends todecrease particularly when the protein is modified to display anantigen.

It is therefore preferable, in order to maintain a sufficient level ofprotein expression in the eukaryotic cell, that the protein (in the caseof serotype y) be modified to retain at least N-terminal amino acidresidues 1 to 20 in the entire amino acid sequence of the pre-S region(pre-S1, pre-S2 regions), or more preferably the protein be modified bydeleting N-terminal amino acids 50 to 153 in the entire amino acidsequence of the pre-S region. For serotype d, the protein is preferablymodified to retain at least N-terminal amino acid residues 12 to 31, ormore preferably the protein is modified by deleting N-terminal aminoacids 61 to 164 in the entire amino acid sequence of the pre-S region.

In this way, the hepatitis B virus surface-antigen protein modified tolose its infectivity to the hepatocytes and display an antibody isexpressed in large amounts in the eukaryotic cell. With the increasedamount of protein, more substance in the protein can be transported intospecific cells or tissues, thereby greatly enhancing the effectivenessof the substance.

An example of the antibody is a cancer specific antibody or ananti-virus protein antibody. For example, a cancer treating substance(medicament) may be encapsulated in the hollow nanoparticles displayinga cancer-specific antibody. This provides an effective therapeutic drugthat specifically and effectively acts on cancer cells. The anti-virusprotein antibody is effective in the removal of virus-infected cells.

The antibody has a single chain or double chain. Due to its structure,the double chain antibody cannot readily be displayed on the particlesurface by directly fusing it with the particle-forming protein. Theinventors of the present invention found ways to successfully displaythe double chain antibody on the surface of the hollow nanoparticles byindirectly binding the double chain antibody to the protein.Specifically, the double chain antibody was displayed on the particlesurface by first introducing a ZZ tag into the protein (fused with theprotein), wherein the ZZ tag specifically binds to the Fc site of thedouble chain antibody, and then by ligating the ZZ tag to the Fc site.Another way to display the double chain antibody on the particle surfaceis to introduce a streptag into the protein (fused with the protein),wherein the streptag specifically binds to streptavidin (or itsderivative), and bind the streptag to the streptavidin (or itsderivative). The double chain antibody, which has been modified bybiotin that specifically binds to the streptavidin (or its derivative),can then be displayed on the particle surface by ligating thestreptavidin (or its derivative) to the biotin attached to the doublechain antibody. The single chain antibody can be displayed on theparticle surface by expressing it with the protein directly fused withthe antibody.

Other than these methods, the antibody may be displayed on the particlesurface by common binding methods involving chemical modification.

The hollow protein nanoparticles are preferably the product ofexpression in eukaryotic cells. The eukaryotic cell may be obtained fromyeasts, insects, or animals including mammals.

The target-cell substance encapsulated in the hollow nanoparticles maybe a cancer treating gene, for example. When the cancer treating geneencapsulated in the drug is a thymidine kinase (HSV1tk) gene derivedfrom simple herpes virus, ganciclovir is additionally administered, aswill be described in Examples.

The present invention discloses a drug that can be used by a convenientmethod of intravenous injection to effectively treat specific diseasedcells or tissues. The drug is a great leap forward from conventionaldisease treatment methods in that it does not require large dose or anysurgical operation in disease treatment including gene therapy, and thatthe risk of side effect is greatly reduced. The drug is therefore usablein clinical applications in its present form.

The present invention discloses a treatment method for treating diseasesthrough administration of the drug disclosed in the present invention.

The present invention discloses hollow nanoparticles of a hepatitis Bvirus surface-antigen protein of serotype y, the hepatitis B virussurface-antigen protein forming particles and being modified to retainat least N-terminal amino acid residues 1 to 20 in the entire amino acidsequence of the pre-S region. Preferably, the protein is modified bydeleting N-terminal amino acids 50 to 153 in the entire amino acidsequence of the pre-S region.

The present invention discloses hollow nanoparticles of a hepatitis Bvirus surface-antigen protein of serotype d, the hepatitis B virussurface-antigen protein forming particles and being modified to retainat least N-terminal amino acid residues 12 to 31 in the entire aminoacid sequence of the pre-S region. Preferably, the protein is modifiedby deleting N-terminal amino acids 61 to 164 in the entire amino acidsequence of the pre-S region.

The hollow nanoparticles are expressed in large amounts particularly inthe eukaryotic cell, and are suitable for displaying bio-recognizingmolecules. For example, the hollow nanoparticles may be used as hollowbio-nanoparticles in gene therapy or DDS.

For a fuller understanding of the nature and advantages of theinvention, reference should be made to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing protein regions of HBsAg genedescribed in Examples of the present invention, where the numbers 1through 8 indicate respective functions of different sites on a surfaceantigen, and Pre-S1 indicates 108 amino acid residues for serotype y,and 119 amino acid residues for serotype d.

FIG. 2 is an explanatory drawing schematically showing one example ofexpression and purification procedures for HBsAg particles usingrecombinant yeasts, as described in Examples of the present invention,wherein (a) illustrates preparation of recombinant yeasts, (b)illustrates incubation in High-Pi medium, (c) illustrates incubation in8S5N-P400 medium, (d) illustrates disruption, (e) illustrates densitygradient centrifugation, and (f) illustrates HBsAg particles.

FIG. 3 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-streptag particles with yeasts, as described inExamples of the present invention.

FIG. 4 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-streptag particles with insect cells, as describedin Examples of the present invention.

FIG. 5 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-streptag particles with animal cells, as describedin Examples of the present invention.

FIG. 6 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-ZZ tag particles with yeasts, as described inExamples of the present invention.

FIG. 7 is a diagram representing results of SDS-PAGE and Westernblotting performed on the HBsAg-ZZ tag particles obtained with yeasts.

FIG. 8 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-ZZ tag particles (or HBsAg-scFv particlesdisplaying single chain antibody A22 or 3A21) with insect cells, asdescribed in Examples of the present invention.

FIG. 9 is a diagram showing steps of constructing a plasmid used forpreparation of HBsAg-ZZ tag particles (or HBsAg-scFv particlesdisplaying single chain antibody A22 or 3A21) with animal cells, asdescribed in Examples of the present invention.

FIG. 10 is a graph showing a result of treatment on laboratory animalsusing the HBsAg-ZZ tag particles as a drug of the present invention.

FIG. 11 is a diagram representing results of SDS-PAGE and Westernblotting performed on the HBsAg-scFv particles.

FIG. 12 is a schematic diagram showing deletion HBsAg protein expressiongenes, as described in Examples of the present invention.

FIG. 13 is a diagram showing reaction compositions of PCR as describedin Examples of the present invention.

FIG. 14 is a diagram showing a PCR cycle as described in Examples of thepresent invention.

FIG. 15 is a schematic diagram illustrating deletion HBsAg proteinexpression genes and a plasmid into which the genes are transferred, asdescribed in Examples of the present invention.

FIGS. 16( a) and 16(b) are graphs showing results of enzyme immunoassayperformed on deletion HBsAg protein in animal cells as described inExamples of the present invention, wherein FIG. 16( a) is a result insupernatant, and FIG. 16( b) is a result in cells.

FIG. 17 is a diagram representing the results of FIGS. 16( a) and 16(b)in data form.

FIG. 18 is a diagram showing results of SDS-PAGE performed on thedeletion HBsAg protein expressed in FIGS. 16( a) and 16(b), wherein (a)is a result in supernatant, and (b) is a result in cells.

FIG. 19 is a diagram showing results of Western blotting performed onthe deletion HBsAg protein expressed in FIGS. 16( a) and 16(b), wherein(a) is a result in supernatant, and (b) is a result in cells.

FIG. 20 is a schematic diagram illustrating deletion HBsAg proteinexpression genes transferred into yeasts, and a plasmid into which thegenes are transferred, as described in Examples of the presentinvention.

FIG. 21 is a diagram showing a result of enzyme immunoassay in dataform, confirming the expression of deletion HBsAg L protein using theplasmid of FIG. 20.

FIG. 22 is a graph representing a result of enzyme immunoassay,confirming the expression of deletion HBsAg L protein using the plasmidof FIG. 20.

FIG. 23 is a diagram listing examples of target-cell substancesaccording to the present invention.

FIG. 24 is a diagram listing examples of target-cell substancesaccording to the present invention.

FIG. 25 is a diagram listing examples of target-cell substancesaccording to the present invention.

FIG. 26 is a diagram listing examples of target-cell substancesaccording to the present invention.

FIG. 27 is a table representing a result of treatment on laboratoryanimals using a drug according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention discloses a drug including hollow nanoparticleswhose particle surface displays an antibody as a bio-recognizingmolecule (molecule that recognizes a specific cell), and which containsparticles encapsulating a substance to be transferred into a cell fortreating a disease, wherein the drug allows the disease-treatingsubstance to be specifically delivered to a target cell or tissue. Thehollow nanoparticles may be a protein able to form particles, which maybe sub viral particles obtained from various viruses. Specific examplesof such a protein include hepatitis B virus (HBV) surface-antigenprotein.

Particles of such a protein may be obtained through the proteinexpression in the eukaryotic cell. Specifically, in eukaryotic cells,the particle-forming protein is expressed on the endoplasmic reticulumas a membrane protein and accumulates thereon before it is released asparticles. The eukaryotic cell may be obtained from yeasts, insects, oranimals including mammals.

As will be described later in Examples, the inventors of the presentinvention have reported that the expression of HBV surface-antigen Lprotein in recombinant yeast cells produces ellipsoidal hollow particleswith a minor axis of 20 nm and a major axis of 150 nm, with a largenumber of L proteins embedded in the yeast-derived lipid bilayermembrane (J. Biol. Chem., Vol. 267, No. 3, 1953-1961, 1992). Theparticles contain no HBV genome and lack the viral function. Therefore,the particles are very safe to the human body.

The HBV surface-antigen L protein may be modified to lack itsinfectivity to the hepatocytes and display an antibody (cancer specificantibody, for example) on the particle surface. With the antibody on theparticle surface of the expressed protein, the protein can effectivelyserve as a carrier for specifically delivering substances to cells ortissues (cancer cell or cancer tissue in the case of a cancer specificantibody) whose cell surface has an antigen against the antibody.

The pre-S regions (pre-S1, pre-S2) of the HBV surface-antigen L proteinhave important roles in the binding of HBV to the hepatocytes (see FIG.1). Thus, the HBV surface-antigen L protein can be modified to lose itsinfectivity to the hepatocyte by deleting some of the amino acids in thepre-S regions. As used herein, “deletion of some of the amino acids inthe pre-S region” means deleting some of the amino acids in the preS1region or preS2 region, or both of these regions. For example,infectivity to the hepatocytes can be lost by deleting N-terminal aminoacids 3 to 66 (serotype y) or N-terminal amino acids 4 to 77 (serotyped), known as a recognition site for the human hepatocytes, in the pre-Sregion (specifically PreS1 region).

When the protein is modified to lose its infectivity to the hepatocytesby deleting at least some of the amino acids in the pre-S region, thelevel of protein expression in the eukaryotic cell varies in themodified hepatitis B virus surface-antigen protein, depending on theregion of amino acid deleted. The level of protein expression is proneto decrease particularly when the protein is modified to displayantigens.

The modified hepatitis B virus surface-antigen protein can be expressedin a large amount in the eukaryotic cell when amino acids in the pre-Sregion are deleted in domain, as will be described in Examples.Specifically, as noted above, the level of protein expression in theeukaryotic cell can be increased by deleting N-terminal amino acids 3 to66 (serotype y) or N-terminal amino acids 4 to 77 (serotype d), known asa recognition site for the human hepatocytes, in the preS1 region. Forserotype y, the protein may be modified to retain at least N-terminalamino acid residues 1 to 20. For serotype d, at least N-terminal aminoacid residues 12 to 31 may be retained.

The level of protein expression can be further increased by preferablydeleting some of the amino acids in the preS2 region, in addition tosome of the amino acids making up the recognition site for the humanhepatocytes in the preS1 region.

More specifically, it is preferable in the entire amino acid sequence inthe pre-S region (pre-S1 region, pre-S2 region) that the protein bemodified to delete N-terminal amino acids 50 to 153 and retain at leastN-terminal amino acid residues 1 to 20. For example, for serotype y, itis preferable in the entire amino acid sequence of the pre-S region thatthe protein be modified to lack domains of amino acid in the first 153amino acids from the N-terminus, specifically, from amino acids 50 to153, 33 to 153, and 21 to 153, as will be described later in Examples.Among these domains, it is particularly preferable to delete amino acids50 to 153. Note that, the deleted range of amino acid is not justlimited to this example.

For serotype d, it is preferable in the entire amino acid sequence ofthe pre-S region (preS1 region, preS2 region) that the protein bemodified to lack N-terminal amino acids 61 to 164 and retain at leastN-terminal amino acid residues 12 to 31.

The hepatitis B virus surface-antigen protein so modified to lose itsinfectivity to the hepatocytes and display antibody is expressed in alarge amount in the eukaryotic cell, and therefore is highlyadvantageous in terms of productivity. With the increased amount ofprotein, more substance in the protein can be transported into specificcells or tissues, thereby greatly enhancing the effectiveness of thesubstance.

Therefore, forming the protein particles using recombinant yeasts offersa preferable method of efficiently producing particles from solubleproteins in the yeasts.

The insect cell, being a eukaryote closer to some of the higher animalsthan the recombinant yeast, is able to form a higher order structuresuch as a sugar chain unachievable by yeasts. In this connection, theinsect cell provides a preferable method of producing heteroproteins inlarge amounts. The conventional insect cell line used the baculovirusand involved viral expression. This has caused a cell death or lysis inthe protein expression. A problem of this method, then, is that theprotein expression proceeds continuously, or the proteins are decomposedby the free protease separated from the dead cells. Further, in thesecretion and expression of proteins, inclusion of a large amount offetal bovine serum contained in the culture medium has made it difficultto purify proteins even when proteins are secreted in the medium. Inrecent years, Invitrogen Corporation has developed and marketed aninsect cell line that can be cultured without a serum and without beingmeditated by the baculovirus. Such an insect line can be used to obtainprotein particles that are easy to purify and form into higher orderstructures.

Hollow protein nanoparticles of the present invention are prepared bybinding an antibody to the surface of particles obtained by theforegoing methods. With various substances (DNA, RNA, proteins,peptides, drugs, etc.) incorporated into the particles, the hollowprotein nanoparticles can very specifically deliver and transfer thesesubstances to cells bearing corresponding antigens on its cell surface.

The particle-forming protein is not just limited to the modifiedhepatitis B virus surface-antigen protein. For example, animal cells,plant cells, viruses, natural proteins derived from fungi, and varioustypes of synthetic proteins may be used. Further, when there is apossibility that, for example, virus-derived antigen proteins maytrigger antibody reaction in a target organism, a particle-formingprotein with suppressed antigenic action may be used. For example, sucha protein may be the hepatitis B virus surface-antigen protein modifiedto suppress its antigenic action, or other types of modified proteins(hepatitis B virus surface-antigen protein modified by geneticengineering), as disclosed in International Publication WO01/64930.

The type of antibody bound to the particle surface is not particularlylimited as long as it recognizes a surface molecule of a specific cellas an antigen. For example, the antibody may be a cancer specificantibody that recognizes a surface molecule of a specific cancer cell asan antigen. As another example, an antibody may be used thatspecifically recognizes an antigen on the surface of a specific cell asa growth factor receptor or cytokine receptor. Other than theseexamples, various types of antibodies specific to other types ofantigens displayed on the cell surface or tissue surface may be used aswell. Specifically, an anti-viral protein antibody may be used, inaddition to the antibodies used in the Examples below. The antibodyshould be suitably selected according to the type of target cell ortissue.

As described, the present invention provides hollow proteinnanoparticles that encapsulate a substance (target-cell substance) to betransferred into a target cell or tissue, and thereby provides asubstance carrier (drug) having cell specificity. The substance carriermay encapsulate any substance including, for example, genes in the formof DNA or RNA, natural or synthetic proteins, oligonucleotides,peptides, drugs, and natural or synthetic compounds.

For example, human RNase1 or RNase3 may be used, as previously reportedby the inventors of the present invention. Human RNase1 is documented inJinno H, Ueda M, Ozawa S, Ikeda T, Enomoto K, Psarras K, Kitajima M,Yamada H, Seno M Life Sci. 1996; 58(21): 1901-8. Human RNase3 (alsoknown as ECP (eosinophil cationic protein)) is documented inMallorqui-Fernandez G, Pous J, Peracaula R, Aymami J, Maeda T, Tada H,Yamada H, Seno M, de Llorens R, Gomis-Ruth F X, Coll M; J Mol Boil. 2000Jul. 28; 300(5): 1297-307.

The proteins have cytotoxicity, the effects of which are bothintracellular and extracellular. With the RNase encapsulated in thesubstance carrier (drug) of the present invention, the cytotoxicity ofthe protein can be masked outside the cell, and the protein exhibits itseffect only inside the cell. It is expected that this will provide anovel cancer treatment method that causes fewer side effects.

Note that, the target-cell substance may be proteins shown in FIG. 23through 26, or genes that encode these proteins. Other examples of thesubstance are various proteins including: cancer suppressor genes (p53,etc.); interferons; interleukins; cytokines; colony stimulating factors;tumor necrosis factors; transforming growth factors β; platelet-derivedgrowth factors; erythropoietins; and Fas antigens. The target-cellsubstance may also be genes that encode these proteins.

These target-cell substances may be incorporated into the hollownanoparticles by various methods commonly used in chemical or molecularbiological experimental techniques. Some of the preferred examplesinclude an electroporation method, ultrasonic method, simple diffusionmethod, and a method using charged lipids.

The hollow protein nanoparticles or substance carrier allow thesubstance to be specifically transported into cells or tissues in vivoor in vitro. Specific transport of the substance into a specific cell orspecific tissue with the use of the hollow protein nanoparticles orsubstance carrier may be used as a treatment method of various diseases,or one of the steps in the procedure of the treatment method.

In a drug according to the present invention, the antibody may bedisplayed on the particle surface by four different methods, as will bedescribed in the Examples. In the first method, a ZZ tag thatspecifically binds to an Fc site of a double chain antibody isincorporated into a particle-forming protein (in other words, particlesare formed by expressing the protein with the ZZ tag fused with theprotein), and the ZZ tag is bound to the Fc site to display the doublechain antibody on the particle surface. In the second method, a streptagthat specifically binds to streptavidin is incorporated into aparticle-forming protein (in other words, particles are formed byexpressing the protein with the streptag fused with the protein). Thestreptag is bound to the streptavidin (or its derivative), which is thenbound to a double chain antibody that has been modified with biotin thatspecifically binds to the streptavidin (or its derivative), therebydisplaying the antibody to the particle surface. In the third method,particles are formed by expressing the particle-forming protein with asingle chain antibody fused with the protein, thereby displaying theantibody on the particle surface. The fourth method is chemical bindingof the antibody with particles with the use of common crosslinkingagents, which may be, for example, compounds including the NHS(N-hydroxysuccinimide) group, maleimide group, or imidoester group(available from Pierce Biotechnology, Inc.). These methods may be partlymodified by taking advantage of their principles.

The effectiveness of the treatment using the drug of the presentinvention has been confirmed by animal testing, as will be describedlater in the Examples. In the Examples, cells derived from humansquamous cell carcinoma were transplanted in nude rats, and the drug ofthe present invention and ganciclovir (GCV) were administered to eachrat in separate doses. The drug on its particle surface had an antibodythat recognizes an antigen, the epidermal growth factor (EGF receptor),expressed by the cancer cells. Inside the drug, a thymidine kinase(HSV1tk) gene derived from simple herpes virus was encapsulated. Theeffectiveness of the treatment was confirmed by observing the size ofgrafted cancer tissue. The drug was administered intravenously. However,oral administration, intramuscular administration, intraperitonealadministration, subcutaneous administration, or other administrationroutes are also available.

In the following, the present invention will be described in more detailby way of Examples with reference to the attached drawings. It should beappreciated that the present invention is not limited in any ways by thefollowing Examples, and various modifications to details of theinvention are possible.

It should also be noted that the techniques described in the followingExamples are all novel and were independently developed by the inventorsof the present invention. The novel techniques include: incorporating aprotein in the preS1 region of the deletion HBV surface-antigen Lprotein; producing a deletion HBV surface-antigen L protein suitable forefficient expression in the eukaryotic cell; incorporating abio-recognizing molecule (antibody) in the deletion HBV surface-antigenL protein for displaying it on the deletion HBV surface-antigen Lprotein; and application of these techniques in gene therapy or DDS.

EXAMPLES

In the following, HBsAg refers to hepatitis B virus surface antigen.HBsAg is an envelope protein of HBV, and includes three kinds ofproteins S, M, and L, as schematically illustrated in FIG. 1. S proteinis an important envelope protein common to all three kinds of proteins.M protein includes the entire sequence of the S protein with additional55 amino acids (pre-S2 peptide) at the N-terminus. L protein containsthe entire sequence of the M protein with additional 108 amino acids(serotype y) or 119 amino acids (serotype d) at the N-terminus. In thefollowing Examples, serotype y was used.

The pre-S regions (pre-S1, pre-S2) of HBV have important roles in thebinding of HBV to the hepatocytes. The Pre-S1 region has a directbinding site for the hepatocytes, and the pre-S2 region has a polymericalbumin receptor that binds to the hepatocytes via polymeric albumin inthe blood.

Expression of HBsAg in the eukaryotic cell causes the protein toaccumulate as membrane protein on the membrane surface of theendoplasmic reticulum. The L protein molecules of HBsAg agglomerate andare released as particles into the ER lumen, carrying the ER membranewith them as they develop.

The Examples below used L proteins of HBsAg. FIG. 2 briefly illustratesprocedures of expression and purification of HBsAg particles describedin the following Examples.

Example A Expression of HBsAg Particles in Recombinant Yeasts

Recombinant yeasts (Saccharomyces cerevisiae AH22R⁻ strain) carrying(pGLDLIIP39-RcT) were cultured in synthetic media High-Pi and 8S5N-P400,and HBsAg L protein particles were expressed (FIG. 2 a through 2 c). Thewhole procedure was performed according to the method described in J.Biol. Chem., Vol. 267, No. 3, 1953-1961, 1992 reported by the inventorsof the present invention.

From the recombinant yeast in stationary growth phase (about 72 hours),the whole cell extract was obtained with the yeast protein extractionreagent (product of Pierce Chemical Co., Ltd.). The sample was thenseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and the HBsAg in the sample was identified by silverstaining.

The result showed that HBsAg was a protein with a molecular weight ofabout 52 kDa.

Example B Purification of HBsAg Particles from the Recombinant Yeasts

(1) The recombinant yeast (wet weight of 26 g) cultured in syntheticmedium 8S5N-P400 was suspended in 100 ml of buffer A (7.5 M urea, 0.1 Msodium phosphate, pH 7.2, 15 mM EDTA, 2 mM PMSF, and 0.1% Tween 80), anddisrupted with glass beads by using a BEAD-BEATER. The supernatant wascollected by centrifugation (FIG. 2 d).

(2) The supernatant was mixed with a 0.75 volume of PEG 6000 solution(33%, w/w), and cooled on ice for 30 min. The pellets were collected bycentrifugation at 7000 rpm for 30 min, and resuspended in buffer Awithout Tween 80.

(3) The solution was layered onto a 10-40% CsCl gradient, andultracentrifuged at 28000 rpm for 16 hours. The centrifuged sample wasdivided into 12 fractions, and each fraction was tested for the presenceof HBsAg by Western blotting (the primary antibody was the anti-HBsAgmonoclonal antibody). The HBsAg fractions were dialyzed against buffer Awithout Tween 80.

(4) 12 ml of the dialyzed solution obtained in (3) was layered onto a5-50% sucrose gradient, and ultracentrifuged at 28000 rpm for 16 hours.As in (3), the centrifuged sample was divided into fractions, and eachfraction was tested for the presence of HBsAg. The HBsAg fractions weredialyzed against buffer A containing 0.85% NaCl, without urea or Tween80 ((2) through (4): FIG. 2 e).

(5) By repeating the procedure (4), the dizlyzed sample was concentratedwith the ultrafilter Q2000 (Advantec), and stored at 4° C. for later use(FIG. 2 f).

The result of Western blotting after CsCl equilibrium centrifugation in(3) revealed that HBsAg was a protein with S antigenicity with amolecular weight of 52 kDa. At the end of the procedure, about 24 mg ofpure HBsAg particles were obtained from the yeast (26 g wet weight)derived from 2.5 L medium.

Each fraction obtained in the purification process was analyzed bySDS-PAGE. In order to confirm whether the purification had successfullyremoved the yeast-derived protease, the HBsAg particles obtained in (5)were incubated at 37° C. for 12 hours, separated by SDS-PAGE, andidentified by silver staining.

The result of confirmation showed that the yeast-derived protease hadbeen completely removed by the purification process.

The HBsAg particles specifically infect the human hepatocytes. Thestrong infectivity of the HBsAg particles is rendered by the hepatocyterecognition site displayed on the particle surface, which has been foundon amino acid residues 3 to 77 in the pre-S1 region (Le Seyec J,Chouteau P, Cannie I, Guguen-Guillouzo C, Gripon P., J. Virol. 1999,March; 73(3): 2052-7).

In the following, description is made as to a producing method of thedrug in which a cancer-specific antibody is displayed on the particlesurface. In the producing method described below, the strong infectivityof the HBsAg particles to the hepatocytes has been removed in order toensure that the drug of the present invention only acts on a specificcancer cell whose cell surface bears a molecule that is recognized as anantigen by the drug antibody. Further, the drug of the present inventionwas prepared in three different forms: (1) HBsAg particles displaying acancer-specific antibody with a streptag; (2) HBsAg particles displayinga cancer-specific antibody with a ZZ tag; and (3) HBsAg particlesdisplaying a cancer-specific single chain antibody expressed with theHBsAg protein fused with the antibody.

Example C Preparation of HBsAg Particles Displaying a Cancer-SpecificAntibody using a Streptag Example C-1 Preparation of HBsAg-StreptagParticles in Yeast Cells

In order to delete a gene region that encodes a human hepatocyterecognition site of the pGLDLIIP39-RcT plasmid discussed in Example Aand at the same time insert a restriction enzyme NotI site (gcggccgc),PCR was run for the pGLDLIIP39-RcT plasmid using the oligonucleotides ofSEQ ID NOs: 1 and 2 as PCR primers. The PCR was carried out with theQuickChange™ Site-Directed Mutagenesis Kit (Stratagene).

Specifically, using Pfu DNA polymerase (Stratagene) as a heat-resistantDNA polymerase, PCR was run in 30 cycles as follows: 30 second denatureat 95° C., 1 minute annealing at 55° C., and 30 minute synthesis at 68°C. The PCR product was treated with restriction enzyme DpnI andtransformed into E. coli DH5α. Then, vector DNA was extracted from theresultant colonies, and the extract was screened for mutantpGLDLIIP39-RcT plasmid based on the base sequence. In the following, theresultant plasmid will be called pGLDLIIP39-RcT-Null plasmid. Note that,in FIG. 3 and the subsequent drawings, a gene region of plasmid encodingHBsAg L protein that lacks the human hepatocyte recognition site will bedenoted by “Null.” For convenience of explanation, such a gene regionwill be called a “Null region.”

In order to add a SacI site and SaII site in the pGLDLIIP39-RcT-Nullplasmid, PCR was run using the oligonucleotides of SEQ ID NOs: 3 and 4as PCR primers, as shown in FIG. 3, wherein the oligonucleotides of SEQID NOs: 3 and 4 had a SacI site and a SalI site, respectively. The PCRamplified the Null region, which contained a promoter (GLDp) and aterminator (PGKt), and cDNA fragments including the Null region wereobtained.

Then, a pRS405+2 μm plasmid, which was prepared by inserting a 2 μmorigin into AatII site of a universal yeast vector pRS405 (Stratagene),was digested with restriction enzymes SacI and SalI. The DNA fragmentsincluding the Null region were then inserted into the cleaved pRS405+2μm plasmid, so as to prepare a pRS405+2 μm-Null plasmid.

Thereafter, synthetic oligonucleotides (oligonucleotide of SEQ ID NO: 5,and oligonucleotide of SEQ ID NO: 6 complementary to SEQ ID NO: 5) thatencode a streptag were annealed and inserted into pRS405+2 μm-Nullplasmid digested with NotI. As a result, a pRS405+2 μm-streptag plasmidwas prepared that included a gene region encoding the streptag. Thestreptag is a peptide that binds to streptavidin with strong affinitylike biotin, and has the sequence (1) SAWRHPQFGG (SEQ ID NO: 27) or (2)WSHPQFEK (SEQ ID NO: 28) from the N-terminus. Sequence (1) functions atthe C-terminus of the protein. The present Examples used the streptag ofsequence (2).

The pRS405+2 μm-streptag plasmid was used to transform yeasts(Saccharomyces cerevisiae AH22R⁻ strain). The resultant transformantswere cultured, and the cultured cells were purified to obtain modifiedHBsAg particles (particles obtained by expressing the streptag fusedwith the HBsAg L protein lacking the human hepatocyte recognition site;hereinafter referred to as HBsAg-streptag particles) according to themethod described in Example B. At the end of the procedure, about 200 μgof pure HBsAg-streptag particles were obtained from the yeasts derivedfrom 1.0 L medium.

Example C-2 Preparation of HBsAg-Streptag Particles in Insect Cells inSerum-Free Medium

Example below describes a producing method of HBsAg-strept-tag particlesusing insect cell lines that can be cultured serum-free without themediation of baculovirus. With the producing method using insect celllines, a higher order structure such as a sugar chain can be realized.

As shown in FIG. 4, PCR was run for the pGLDLIIP39-RcT-Null plasmidobtained in Example C-1, using the oligonucleotides of SEQ ID NO: 7 andSEQ ID NO: 8 as PCR primers, wherein the oligonucleotides of SEQ ID NO:7 and SEQ ID NO: 8 had a kpni site (ggtacc) and a SacII site (ccgcgg),respectively. The PCR amplified the Null region, which contained acoding region for a lysozym-secreted signal peptide derived from chicks.

The PCR product was electrophorased on agarose, and gene fragments of atarget band about 1.3 kbp were collected. The gene fragment was ligatedbetween the kpni site and SacII site of vector pIZT/V5-His (used forstable expression in insect cells) (Invitrogen Corporation) to close thering, using TaKaRa Ligation kit ver. 2 (TaKaRa). The base sequence wasconfirmed, and the plasmid was named pIZT-Null.

Thereafter, as in Example C-1, synthetic oligonucleotides(oligonucleotide of SEQ ID NO: 5, and oligonucleotide of SEQ ID NO: 6complementary to SEQ ID NO: 5) that encodes a streptag were annealed andinserted into the pIZT-Null plasmid digested with NotI. As a result, apIZT-streptag plasmid was prepared that included a gene region encodingthe streptag.

Meanwhile, the insect cell High Five line (BTI-TN-5B1-4): (InvitrogenCorporation) was slowly conditioned from the fetal bovineserum-contained medium to a serum-free medium (Ultimate InsectSerum-Free Medium: Invitrogen Corporation) over a period of about 1month. Then, using the gene transfer lipid Insectin-Plus (InvitrogenCorporation), the pIZT-streptag plasmid was transferred for thetransformation of the High Five line conditioned to the serum-freemedium. The sample was incubated in the serum-free medium at 27° C. for48 hours, followed by further incubation that extended 4 to 7 days untilconfluent cells were obtained on the serum-free medium with theadditional 400 μg/mL antibiotic zeocin (Invitrogen Corporation). As aresult, HBsAg-streptag particles were obtained.

The sample was centrifuged at 1500×g for 5 min, and the supernatant wascollected. The HBsAg-streptag particles in the medium were measured forthe presence or absence of expression, using the IMx kit (Dainabot Co.Ltd.). The result confirmed the expression of HBsAg-streptag particles.The HBsAg-streptag particles obtained from the supernatant wereseparated by SDS-PAGE and analyzed by Western blotting using an anti-Santibody (prepared by the inventors), followed by enzyme immunoassayIMx. The HBsAg-streptag particles fused with the streptag had amolecular weight of about 42 kDa.

1 L of the supernatant was concentrated with an ultrafiltration unit(filter UK-200, the product of Advantec, exclusion molecular weight 200K), and purified through an anion exchange column (DEAE-Toyopearl 650 M,Toyo Soda). As a result, about 1 mg of pure uniform HBsAg-streptagparticles were obtained.

Example C-3 Preparation of HBsAg-Streptag Particles in Animal Cells

As shown in FIG. 5, restriction enzyme XhoI was used to cleave thepGLDLIIP39-RcT-Null plasmid at the Xho site, so as to obtain fragmentscontaining the Null region with a terminator (PGKt). After digestingpcDNA3.1 (Invitrogen Corporation) with restriction enzyme XhoI, thefragments were inserted into the pcDNA3.1 to prepare a pcDNA3.1-Nullplasmid.

Thereafter, as in Example C-1, synthetic oligonucleotides(oligonucleotide of SEQ ID NO: 5, and oligonucleotide of SEQ ID NO: 6complementary to SEQ ID NO: 5) that encodes a streptag gene wereannealed and inserted into the pcDNA3.1-Null plasmid digested with NotI.As a result, a pcDNA3.1-streptag plasmid was prepared that included acoding region for the streptag.

The pcDNA3.1-streptag plasmid so obtained was then transferred into COS7cells derived from the monkey kidney, using the gene transfer devicegene pulser (Bio-Rad Laboratories, Inc.). After the transfer, the samplewas incubated overnight in a Dulbecco-modified medium containing 10%fetal bovine serum. After further incubation in a serum-free mediumCHO-SFMII (Gibco-BRL) for a week, the medium was purified to obtainHBsAg-streptag particles.

As in Example C-2, the HBsAg-streptag particles obtained from thesupernatant were separated by SDS-PAGE and analyzed by Western blottingusing an anti-S antibody, followed by enzyme immunoassay IMx. TheHBsAg-streptag particles fused with the streptag had a molecular weightof about 42 kDa. The measured values of IMx were 8.81 (against cut-offvalue) for the wild-type HBsAg L particles expressed with the pcDNA3.1vector, 3.47 for the HBsAg Null particles, and 2.41 for theHBsAg-streptag particles. All of these values can be considered to besufficient.

Example C-4 Method of Displaying an Antibody on the HBsAg-StreptagParticles having a Streptag

The foregoing Examples C-1 through C-3 prepared HBsAg-streptag particleswith a streptag. The streptag specifically binds to streptavidin, whichin turn specifically binds to biotin. By taking advantage of thesespecific bindings, the HBsAg-streptag particles are first bound tostreptavidin, which is then ligated to a biotin-modified antibody. Theresult is HBsAg-streptag particles with the antibodies arrayed on theparticle surface (such HBsAg-streptag particles will be referred to as“HBsAg-streptag-Ab particles” hereinafter).

Specifically, the anti-human EGFR mouse monoclonal antibody 7G7B6(purified), which is an antibody against the human epidermal growthfactor receptor (EGFR), is used as an antibody, and the NHS-biotin(EZ-Link® NHS-Biotin, the product of Pierce Biotechnology, Inc.) wastagged according to the protocol described in the instructions of thePierce product. The purified HBsAg-streptag particles were then ligatedto the avidin protein (ImmunoPure Avidin, the product of PierceBiotechnology, Inc.) by mixing the two in PBS at a molar ratio of 2:1and at ordinary temperature for 30 min (molar calculation was made on amolecular basis). Thereafter, the biotin-tagged anti-human EGFR mousemonoclonal antibody was allowed to react with an equimolar amount ofHBsAg-streptag particles bearing the avidin protein. The reaction wascarried out in PBS at ordinary temperature for 30 min. The result wasHBsAg-streptag-Ab particles bearing the antibodies on the particlesurface.

Example C-5 Transfer of Genes into the HBsAg-Streptag-Ab Particles

According to the method described in International PublicationWO01/64930, the HBsAg-streptag-Ab particles were mixed with a greenfluorescent protein expression plasmid (pEGFP-F (Clontech)), and thepEGFP-F was sealed in the HBsAg-streptag-Ab particles by anelectroporation method. The result was HBsAg-streptag-Ab particles thathad anti-human EGFR antibodies on the particle surface and encapsulatedGFP expression plasmid inside the particles.

Next, there were prepared human squamous cell carcinoma-derived cellsA431 (JCRB9009), along with human hepatic cancer-derived cells NUE andhuman colon cancer-derived cells WiDr as negative controls. The A431 andnegative controls (NUE, WiDr) were each placed on a 3.5 cmglass-bottomed Petri dish, and incubated for 4 days with 1 μg ofHBsAg-streptag-Ab particles encapsulating the GFP expression vectorplasmid. GFP expression in the cells of the respective samples wereobserved with a confocal laser fluorescence microscope.

The observation found GFP fluorescence in A431 but not in the negativecontrols (NUE cells, WiDr).

Thus, with the HBsAg-streptag-Ab particles that had anti-human EGFRantibodies on the particle surface and encapsulated GFP expressionplasmid inside the particles, the experiment showed that the transferand expression of the gene was very specific and efficient in the A431cells on the cultured cell level. The experiment therefore suggests thatthe HBsAg-streptag-Ab particles encapsulating a substance to betransferred into a cell for treating a disease have a potential use inthe effective treatment of specific diseased cells or tissues.

Example D Preparation of Antibody-Displaying HBsAg-ZZ Particles Using aZZ Tag Example D-1 Preparation of HBsAg-ZZ Particles in Yeast Cells

As in Example C-1, a pRS405+2 μm-Null plasmid was prepared as shown inFIG. 6.

Using NotI site-containing oligonucleotides of SEQ ID NOs: 9 and 10 asPCR primers, PCR was run for a plasmid that contained a coding regionfor a ZZ tag (indicated by “ZZ” in the figure; hereinafter referred toas “ZZ region”) (prepared by inserting a ZZ region based on a Protein Agene derived from Staphylococcus aureus). The PCR amplified regionsincluding the ZZ region. The ZZ tag is defined as an amino acid sequencewith the ability to bind to the Fc region of immunoglobulin G, whereinthe amino acid sequence has the following two repeating units from theN-terminus (ZZ tag sequence (SEQ ID NO: 29)):

VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNDAQAPK

The pRS405+2 μm-Null plasmid was then digested with restriction enzymeNotI, and the amplified fragments were inserted in the cleaved plasmidto prepare a pRS405+2 μm-ZZ plasmid.

As in Example A, the plasmid gene pRS405+2 μm-ZZ was used to transformthe yeast S. cerevisiae AH22R⁻ by a spheroplast method. The resultingtransformants were incubated in medium High-Pi (3 ml) at 30° C. for 3days, and subsequently in medium 8S5N-P400 (3 ml) at 30° C. for another3 days, so as to prepare HBsAg particles displaying a ZZ tag.

The pRS405+2 μm-ZZ plasmid so obtained was used to transform the yeast(Saccharomyces cerevisiae AH22R⁻ strain). The resulting transformantswere incubated, and the cultured cells were purified according to themethod described in Example B to obtain modified HBsAg particles(particles obtained by expressing the ZZ tag fused with the HBsAg Lprotein lacking the human hepatocyte recognition site). The efficiencyof particle expression was very high, producing about 20 mg of pureHBsAg-ZZ tag particles from the yeasts derived from 1.0 L medium.

The HBsAg-ZZ tag particles obtained from the supernatant were separatedby SDS-PAGE, and analyzed by Western blotting using an anti-S antibody,followed by enzyme immunoassay IMx. FIG. 7 shows the results of SDS-PAGEand Western blotting. The measured values of IMx were 49.43 (againstcut-off value, ×100 diluted) for the wild-type HBsAg L particlesexpressed with the pRS405+2 μm vector, 21.87 for the HBsAg Nullparticles, and 253.64 for the HBsAg-ZZ tag particles. All of thesevalues can be considered to be sufficient. The HBsAg-ZZ tag particleswith the ZZ tag had a molecular weight of about 56 kDa.

Example D-2 Preparation of HBsAg-ZZ Tag Particles in Insect Cells inSerum-Free Medium

As shown in FIG. 8, a pIZT-Null plasmid was obtained according to themethod described in Example C-2.

A region including the ZZ region was inserted in the pIZT-Null plasmidaccording to the method of Example D-1, so as to prepare a pIZT-ZZplasmid.

The pIZT-ZZ plasmid so obtained was inserted in insect cells, andHBsAg-ZZ tag particles were expressed therein according to the method ofExample C-2.

After incubating the insect cells, the supernatant was collected. TheHBsAg-ZZ tag particles obtained from the supernatant were separated bythe SDS-PAGE, and analyzed by Western blotting using an anti-S antibody(prepared by the inventors, mouse polyclonal antibody). The resultshowed that the HBsAg-ZZ tag particles displaying the ZZ tag had amolecular weight of about 56 kDa.

The amount of HBsAg-ZZ tag particles obtained from 1 L supernatant wasabout 1 mg according to the method of Example C-2.

Example D-3 Preparation of HBsAg-ZZ Tag Particles in Animal Cells

As shown in FIG. 9, a pcDNA3.1-Null plasmid was obtained according tothe method described in Example C-3.

Then, a region including the ZZ region was inserted in the pcDNA3.1-Nullplasmid according to the method of Example D-1, so as to prepare apcDNA3.1-ZZ plasmid.

The pcDNA3.1-ZZ plasmid so obtained was inserted in COS7 cells, andHBsAg-ZZ tag particles were expressed therein according to the method ofExample C-3.

After incubating the COS7 cells, the supernatant was collected. TheHBsAg-ZZ tag particles obtained from the supernatant were separated bythe SDS-PAGE, and analyzed by Western blotting using an anti-S antibody(prepared by the inventors, mouse polyclonal antibody), followed byenzyme immunoassay IMx. The measured values of IMx were 8.81 (againstcut-off value) for the wild-type HBsAg L particles expressed with thepcDNA3.1 vector, 3.47 for the HBsAg Null particles, and 2.41 for theHBsAg-ZZ tag particles. All of these values can be considered to besufficient. The HBsAg-ZZ tag particles with the ZZ tag had a molecularweight of about 56 kDa.

Example D-4 Method of Displaying an Antibody on the HBsAg-ZZ TagParticles

The ZZ tag has strong affinity to the Fc portion of the antibodymolecule. For example, the ZZ tag can specifically bind to thecancer-specific mouse monoclonal antibody 7G7B6 against the human EGFreceptor (EGFR). Other examples of antibodies specific to the ZZ taginclude the mouse monoclonal antibody 528 against the human IL-2receptor (Tac antigen), and the colon cancer-specific mouse monoclonalantibody ST-421 against the human colon cancer. By binding the HBsAg-ZZtag particles to these antibodies, HBsAg-ZZ tag particles were preparedthat had the antibodies arrayed on the particle surface (hereinafter,such HBsAg-ZZ tag particles will be referred to as “HBsAg-ZZ tag-Abparticles”).

Specifically, the HBsAg-ZZ tag particles were mixed with an equimolaramount of anti-human EGFR mouse monoclonal antibody 7G7B6 (purified)(molar calculation was made on a molecular basis), and the mixture wasallowed to react for one hour in PBS. The result was HBsAg-ZZ tag-Abparticles with the antibodies displayed on the particle surface.

Example D-5 Transfer of Genes into the HBsAg-ZZ Tag-Ab Particles

According to the method disclosed in International PublicationWO01/64930, the HBsAg-ZZ tag-Ab particles were mixed with a greenfluorescent protein expression plasmid (pEGFP-F (Clontech)), and thepEGFP-F was sealed in the HBsAg-ZZ tag-Ab particles by anelectroporation method. The result was HBsAg-ZZ tag-Ab particles thathad anti-human EGFR antibodies on the particle surface and encapsulatedGFP expression plasmid inside the particles.

Next, as in Example C-5, there were prepared human squamous cellcarcinoma-derived cells A431, along with human hepatic cancer-derivedcells NUE and HuH-7 (JCRB0403) and human colon cancer-derived cells WiDr(ATCC CCL-218) as negative controls. The A431 and negative controls(NUE, HuH-7, WiDr) were each placed on a 3.5 cm glass-bottomed Petridish, and incubated for 4 days with 1 μg of HBsAg-ZZtag-Ab-GFP particlesencapsulating the GFP expression vector plasmid. GFP expression in thecells of the respective samples were observed with a confocal laserfluorescence microscope.

The observation found GFP fluorescence in A431 but not in the othercells (e.g., NUE cells).

Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human EGFRantibodies on the particle surface and encapsulated GFP expressionplasmid inside the particles, the experiment showed that the transferand expression of the gene was very specific and efficient in the A431cells on the cultured cell level.

Meanwhile, human tumor strains (A431, HuH-7, WiDr) were injected byhypodermic injection into nude mice (lineage: BALB/c, nu/nu,microbiological quality: SPF, male, 5 weeks of age). The injection wasmade in the bilateral dorsal area of the mouse with 1×10⁷ cells for eachstrain. In order to obtain a carrier mice, the mice were grown for 2 to4 weeks until the transplanted tumor developed into a solid cancer tumorof about 2 cm diameter.

The HBsAg-ZZ tag-Ab particles encapsulating the GFP expression plasmidwere administered into the abdomen of each mouse with a 26 G syringe.The mouse was killed 4 days after the administration, and the tumor areawas removed along with various organs including liver, spleen, kidney,and intestines. The tissues were fixed and embedded using the GFP resinembedding kit (Technovit 7100).

Specifically, the samples were fixed by immersing them in 4% neutralizedformaldehyde, and were dried in 70% EtOH at room temperature for 2hours, 96% EtOH at room temperature for 2 hours, and 100% EtOH at roomtemperature for one hour. Pre-fixation was carried out for 2 hours atroom temperature in a mixture containing equal amounts of 100% EtOH andTechnovit 7100. The samples were further immersed in Technovit 7100 forno longer than 24 hours at room temperature. Out of the solution, thesamples were allowed to stand for one hour at room temperature and foranother one hour at 37° C. for polymerization.

According to ordinary method, the sample were sliced and stained withhematoxin-eosin (common method of tissue staining). GFP fluorescence ofeach slice was observed with a fluorescent microscope. The result showedthat human squamous cell carcinoma-derived cells A431 had GFPfluorescence. No fluorescence was observed in the organs removed fromthe same mouse, including liver, spleen, kidney, and intestines. On theother hand, in carrier mice that have incorporated cells derived fromother types of human cancer (HuH-7, WiDr), no GFP fluorescence wasobserved in the tumor area, or in the liver, spleen, kidney, orintestines. Fluorescence was not observed either in carrier mice towhich the HBsAg-ZZ tag was not administered.

Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human EGFRantibodies on the particle surface and encapsulated GFP expressionplasmid inside the particles, the experiment showed that the transferand expression of the gene was very specific and efficient in the A431cells on the laboratory animal level.

Example D-6 Effectiveness of Treatment using the HBsAg-ZZ Tag-AbParticles

In order to produce the HBsAg-ZZ tag-Ab particles as a drug of thepresent invention encapsulating HSV1tk gene, a cancer-treating thymidinekinase derived from simple herpes virus (HSV1tk) was sealed in theHBsAg-ZZ tag-Ab particles that were prepared in yeasts according to thedescribed method.

The cancer cells that have incorporated the HSV1tk gene becomeganciclovir (GCV) sensitive when they express the gene. Administrationof ganciclovir therefore kills off the cancer cells by the strong effectit exhibits on the cancer cells. This is one reason the HSV1tk gene hasbeen widely used in the gene therapy of cancer.

In this Example, the HSV1tk gene was sealed in the HBsAg-ZZ tag-Abparticles using a vector pGT65-hIFN-α (the product of InvitrogenCorporation) that expresses the HSV1tk gene. The HBsAg-ZZ tag-Abparticles encapsulating the HSV1tk gene were obtained by transferringthe expression vector into the HBsAg-ZZ tag-Ab particles by anelectroporation method. Specifically, 10 μg of expression vector wastransferred into 50 μg of L protein particles in the HBsAg-ZZ tag-Abparticles. The vector was transferred using a PBS buffer, and theelectroporation was carried out with a 4 mm cuvette under 220 V and 950μF.

As the laboratory animal, the present Example used nude rats purchasedfrom CLEA Japan, Inc. (lineage: F344/NJcl-rnu/rnu, female). Byhypodermic injection, human squamous cell carcinoma-derived cells A431were transplanted into the nude rats, along with the human coloncancer-derived cells WiDr as a negative control. The injection was madein the bilateral dorsal area of the rats with 1×10⁷ cells for each celltype. The rats were grown for about 3 weeks until the grafted tumordeveloped into a solid cancer tumor of about 2 to 3 cm diameter.

10 μg of HBsAg-ZZ tag-Ab particles encapsulating the HSV1tk gene wereadministered to each nude rat through the tail vein (intravenousinjection). Starting from 5 days after the intravenous injection,ganciclovir (GCV) was administered to each rat with the dose of 50mg/kg/day, using an osmotic pump (alzet osmotic pump; Cat No. 2ML2).Here, the GCV was administered to the back of each nude ratsubcutaneously. The GCV was administered for no longer than 14 days.After the administration, the state (size) of the tumor tissue of thenude rats was observed over time. Specifically, the major axis and minoraxis of the tumor part were measured with a gauge, and a tumor volumewas approximated (major axis×minor axis×minor axis/2). The rats weremeasured in triplet. The results are shown in FIG. 10 and FIG. 27.

Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human EGFRantibodies on the particle surface and encapsulated the HSV1tk geneinside the particles, the experiment showed that the transfer andexpression of the gene was very specific and efficient in the A431 cellsand therefore highly effective in cancer treatment on the laboratoryanimal level.

Example E Preparation of HBsAg-scFv Particles Displaying a Single ChainAntibody Example E-1 Preparation of HBsAg-scFv Particles in Yeast Cells

A region of including a coding region for antibody A22 or 3A21 wasamplified by PCR, where the antibody A22 is a single chain anti-humanserum albumin antibody derived from mice, and the antibody 3A21 is asingle chain anti-human RNase antibody derived from mice. PCR wasperformed according to the procedure of Example D-1, except that adifferent plasmid and different PCR primers were used. As the plasmidthat includes a ZZ region, the present Example used either a plasmidthat includes a coding region for the antibody A22 (generous gift ofTOTO LTD.), or a plasmid that includes a coding region for the antibody3A21. (prepared according to the method described in Mol Immunol. 1997August-September; 34(12-13): 887-90 Katakura Y, Kumamoto T, Iwai Y,Kurokawa Y, Omasa T, Suga K., and Mol Immunol 1997 July; 34(10): 731-4Katakura Y, Kumamoto T, Iwai Y, Kurokawa Y, Omasa T, Suga K.) As the PCRprimers, the present Example used either oligonucleotides of SEQ ID NOs:11 and 12 (in the case of A22), or oligonucleotides of SEQ ID NOs: 13and 14 (in the case of 3A21), where each oligonucleotide had a NotIsite. The single chain antibody (scFv) is a pseudo antibody moleculethat has been restructured to have the antigen recognition site only ona single chain polypeptide, rather than the normal double chainpolypeptide.

The amplified fragments obtained by the PCR were inserted in thepRS405+2 μm-Null plasmid to prepare pRS405+2 μm-A22 plasmid or pRS405+2μm-3A21 plasmid. The pRS405+2 μm-A22 or pRS405+2 μm-3A21 plasmid wastransferred into yeasts and expressed therein. The result was particleswhose particle surface had single chain antibody A22 or 3A21 expressedwith the HBsAg L protein fused with the antibody (such particles will bereferred to as HBsAg-scFv particles hereinafter).

After incubating the yeasts, the supernatant was collected. TheHBsAg-scFv particles obtained from the supernatant were separated bySDS-PAGE, and analyzed by Western blotting using an anti-S antibody,followed by enzyme immunoassay IMx. FIG. 11 shows the results ofSDS-PAGE and Western blotting. The measured values of IMx were 49.43(against cut-off value, ×100 diluted) for the wild-type HBsAg Lparticles expressed with the pRS405+2 μm vector, 21.87 for the HBsAgNull particles, 2.41 for the HBsAg-scFv particles displaying A22, and4.02 for the HBsAg-scFv particles displaying 3A21. All of these valuescan be considered to be sufficient. The HBsAg-scFv particles withantibody A22 had a molecular weight of about 76 kDa. The result was thesame for the HBsAg-scFv particles with antibody 3A21. By the method ofExample C-2, about 200 μg of pure HBsAg-scFv particles were obtainedfrom the yeasts derived from 1.0 L medium.

Example E-2 Preparation of HBsAg-scFv Particles in Insect Cells inSerum-Free Medium

As shown in FIG. 8, a region of including a coding region for antibodyA22 or 3A21 was amplified by PCR, where the antibody A22 is a singlechain anti-human serum albumin antibody derived from mice, and theantibody 3A21 is a single chain anti-human RNase antibody derived frommice. PCR was performed according to the procedure of Example D-2,except that a different plasmid and different PCR primers were used. Asthe plasmid that includes a ZZ region, a plasmid that includes a codingregion for the antibody A22 or 3A21 was used. As the PCR primers, thepresent Example used either oligonucleotides of SEQ ID NOs: 11 and 12(in the case of A22), or oligonucleotides of SEQ ID NOs: 13 and 14 (inthe case of 3A21), where each oligonucleotide had a NotI site.

The amplified fragments obtained by the PCR were inserted in thepRS405+2 μm-Null plasmid to prepare pIZT-A22 plasmid or pIZT-3A21plasmid. The pIZT-A22 or pIZT-3A21 plasmid was transferred into insectcells and expressed therein. The result was HBsAg-scFv particlesdisplaying the single chain antibody A22 or 3A21.

After incubating the insect cells, the supernatant was collected. TheHBsAg-scFv particles obtained from the supernatant were separated bySDS-PAGE, and analyzed by Western blotting using an anti-S antibody. TheHBsAg-scFv particles with antibody A22 had a molecular weight of about76 kDa. The result was the same for the HBsAg-scFv particles withantibody 3A21.

By the method of Example C-2, about 1 mg of pure HBsAg-scFv particleswere obtained from 1.0 L supernatant.

Example E-3 Preparation of HBsAg-scFv Particles in Animal Cells

As shown in FIG. 9, a region including a coding region for antibody A22or 3A21 was amplified by PCR, where the antibody A22 is a single chainanti-human serum albumin antibody derived from mice, and the antibody3A21 is a single chain anti-human RNase antibody derived from mice. PCRwas performed according to the procedure of Example D-3, except that adifferent plasmid and different PCR primers were used. As the plasmidthat includes a ZZ region, a plasmid that includes a coding region forthe antibody A22 or 3A21 was used. As the PCR primers, the presentExample used either oligonucleotides of SEQ ID NOs: 11 and 12 (in thecase of A22), or oligonucleotides of SEQ ID NOs: 13 and 14 (in the caseof 3A21), where each oligonucleotide had a NotI site.

The amplified fragments obtained by the PCR were inserted in thepcDNA3.1 plasmid to prepare pcDNA3.1-A22 plasmid or pcDNA3.1-3A21plasmid. The pcDNA3.1-A22 or pcDNA3.1-3A21 plasmid was transferred intoanimal cells and expressed therein. The result was HBsAg-scFv particlesdisplaying the single chain antibody A22 or 3A21.

After incubating the COS7 cells, the supernatant was collected. TheHBsAg-scFv particles obtained from the supernatant were separated bySDS-PAGE, and analyzed by Western blotting using an anti-S antibody. Theresult showed that HBsAg-scFv particles with antibody A22 had amolecular weight of about 76 kDa. The result was the same for theHBsAg-scFv particles with antibody 3A21.

Example E-4 Transfer of Genes into the HBsAg-scFv Particles

The HBsAg-scFv particles so prepared were fixed on 96-well plates,wherein human serum albumin was used for the HBsAg-scFv particles thathad antibody A22, and human RNase 1 was used for the HBsAg-scFvparticles that had 3A21 antibody. Binding factors of the respectivesamples were measured by an ELISA. The amount of HBsAg-scFv particlesthat bound in the stationary phase was quantified using the HRP taganti-HBsAg polyclonal antibody provided in the AUSZYME II of DainabotCo. Ltd. The result showed that the HBsAg-scFv particles had bindingfactors of not more than 100 nM for A22, and not more than 50 nM for3A21. The results are based on proteins building the HBsAg-scFvparticles, not the HBsAg-scFv particles themselves. The binding factorsin these ranges are sufficient for the HBsAg-scFv particles to serve asa carrier for delivering a drug or other substances to a specific siteinside the body.

The experiment showed that the HBsAg-scFv particles displaying theantibody A22 or 3A21 on the particle surface were highly specific to theA431 cells.

Example F

By expressing various types of deletion HBsAg L proteins lacking aminoacids in the pre-S region (pre-S1, pre-S2), which is the humanhepatocyte recognition site of the HBsAg L protein, the present Exampleevaluated the level of expression and antigenicity in eukaryotic cellsamong different amino acid deletion regions.

Example F-1 Preparation of Deletion HBsAg L Protein Expression Genes

Deletion HBsAg L protein expression genes were prepared by PCR accordingto the method described below.

In order to obtain deletion HBsAg L proteins, there were prepareddeletion HBsAg L protein expression genes that express 5 types ofdeletion HBsAg L proteins (a) to (e) below in which part of the pre-Sregions (pre-S1 region, pre-S2 region) has been deleted. Specifically,the deletion HBsAg L proteins prepared in this Example are (a) a proteinlacking N-terminal amino acids 21 to 153 in the pre-S region (Δ21-153 inFIG. 12; the same notation is used below), (b) a protein lackingN-terminal amino acids 33 to 153 in the pre-S region (Δ33-153), (c) aprotein lacking N-terminal amino acids 50 to 153 in the pre-S region(Δ50-153), (d) a protein lacking N-terminal amino acids 108 to 153 inthe pre-S region (Δ108-153), and (e) a protein lacking N-terminal aminoacids 127 to 153 in the pre-S region (Δ127-153).

In order to amplify deletion HBsAg L protein expression genes of therespective proteins (a) through (e), PCR was run for pB0477 (plasmidthat has incorporated HbsAg L protein expression genes, prepared by theinventors) according to the described method. As the PCR primers, theoligonucleotides of SEQ ID NOs: 15 through 24 were used. Theoligonucleotides of SEQ ID NOs: 15 and 16 were for amplifying thedeletion HBsAg L protein (a), the oligonucleotides of SEQ ID NOs: 17 and18 for (b), the oligonucleotides of SEQ ID NOs: 19 and 20 for (c), theoligonucleotides of SEQ ID NOs: 21 and 22 for (d), and theoligonucleotides of SEQ ID NOs: 23 and 24 for (e). Further, among theprimers of SEQ ID NOs: 15 through 24, the odd-numbered ones are forwardprimers, and the even-numbered ones are reverse primers.

The reaction compositions of PCR are shown in FIG., 13: Pyrobest DNApolymerase (TaKaRa) (heat-resistant DNA polymerase) (0.5 μL), PCR buffer(5 μL×10), dNTP mixture (10 mM, 5 μL), template DNA (pB0477; plasmidthat has incorporated the HbsAg L protein expression genes, prepared bythe inventors) (5 μg/mL, 2 μL), and a primer set (SEQ ID NOs: 15 to 24)(1 μL each). The total volume was 50 μL with the addition of distilledwater.

The PCR was run in 30 cycles as follows: 30 second denature at 98° C.,30 second denature at 98° C., 1 minute annealing at 55° C., and 30minute synthesis at 68° C. The reaction was ended upon cooling to 4° C.,as shown in FIG. 14. In order to cut the template DNA, the restrictionenzyme DpnI (1OU) was added to the PCR product. After incubation at 37°C. for 1 hour, the resulting plasmid was used to transform E. coli JM109strain. The plasmid was removed from the expression colonies, and itsbase sequence was confirmed.

Thereafter, restriction enzyme NotI sites were introduced into thedeletion HBsAg L protein. FIG. 15 schematically illustrates anexpression gene that was prepared by introducing restriction enzyme NotIsites in the deletion HBsAg L protein expression gene. The schematicdiagram of FIG. 15 also illustrates a plasmid that has incorporated sucha gene. In FIG. 15, the restriction enzyme NotI sites are indicated by0aa, 25aa, and ΔPreS, wherein 0aa is an insertion site at an end (5′end) of the deletion HBsAg L protein expression gene, 25aa is aninsertion site at the 3′ end of the first 25 amino acid residues fromthe 5′ end, and ΔPreS is an insertion site at an end (5′ end) of an Sprotein expression gene.

The deletion HBsAg L protein expression gene with the NotI sites wasthen inserted in a plasmid pB0477 (plasmid that has incorporated theHBsAg L protein expression gene, for expression in animal cells,prepared by the inventors) with XhoI, so as to obtain a recombinantHBsAg L protein expression gene.

Note that, in FIG. 15 and subsequent drawings, the notation A127-153indicates that the HBsAg L protein expression gene shown in FIG. 12lacks a gene that encodes amino acids 127 to 153, for example. (The samenotation is used below.) Similarly, Δpre-S indicates that a gene thatencodes all amino acids in the pre-S regions (pre-S1, pre-S2) islacking.

Example F-2 Preparation of Deletion HBsAg L Protein in Animal Cells

The plasmid (2 μg) constructed in Example F-1 was used to transform Cos7cells (3 to 8×10⁴ cells) by electroporation (300 V, 950 μF). Theresulting plasmid was allowed to stand at 37° C. for 4 days in thepresence of 5% CO₂. The amount of mutant L particles (deletion HBsAg Lprotein) in the supernatant and cell extract was measured with an enzymeimmunoassay device (Dainabot Co. Ltd.). The measurement was made basedon the antigenicity of the mutant L particles. The supernatant used inthe measurement had been diluted with the equal amount of PBS. The cellextract was obtained by causing the cells to lyse in a lysis buffer (20mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 10 mM 2-mercaptoethanol, 1% (v/v)Triton X-100), followed by ×200 dilution of the lysate supernatant withPBS after centrifugation.

FIGS. 16( a) and 16(b) and FIG. 17 represent the results of measurement,showing the produced amount of mutant L particles (given in numericalvalues and graph). In these drawings, greater values of S/N and RATEindicate greater antigenicity. That is, samples Δ21-153, Δ33-153, andΔ50-153 produced good results, of which the deletion HBsAg L proteinΔ50-153 was particularly desirable.

The level of expression was also measured by SDS-PAGE and Westernblotting, as shown in FIG. 18 and FIG. 19. As the primary antibody, amouse anti-S protein antibody (prepared by the inventors) was used. Theanti-mouse IgG antibody AP tag (Promega) was used as the secondaryantibody. Note that, FIG. 19 shows the result of Western blotting afterenzyme treatment (EndH), which was performed to remove N-sugar chains.The result is shown along with the molecular weights. The EndH treatmentrevealed that the Pre-S region of the product mutant L particles hadN-sugar chains. For Δ51-66 in FIG. 19, a plasmid prepared with theprimers of SEQ ID NOs: 25 and 26 were used according to the methoddescribed in Example F-1.

The experiment showed that the level of protein expression wasparticularly desirable in the deletion HBsAg L proteins (a) through (c).

Example F-3 Preparation of Deletion HBsAg L Proteins with InsertedEpithelial Growth Factor (EGF)

Using the deletion HBsAg L protein expression genes (a) through (c)(Δ21-153, Δ33-153, and Δ50-153) which showed desirable levels of proteinexpression, the EGF gene was inserted in these genes at the NotI sitesand expressed therein. The EGF gene was obtained by cleaving thepGLDLIIP39-RcT-EGF (prepared by the inventors) with the restrictionenzyme NotI. The resulting plasmid was used to transform the Cos7 cells.After 24 hr incubation in serum media, the samples were furtherincubated for 3 days on serum-free media. The culture media werecollected and concentrated with an ultrafilter, so as to obtain mutant Lparticles (deletion HBsAg L proteins (a) through (c)).

The green fluorescent protein expression plasmid (pEGFP-F (Clontech))was electroporated in the particles of the respective proteins, and theGFP expression plasmid was encapsulated in the particles. The resultingparticles were used in a gene transfer experiment using hepatocyte HepG2and epithelial cell A431. Observation of the GFP fluorescence showedthat specificity to the hepatocyte HepG2 had been lost, and that bindingto the epithelial cell A431 was highly selective. That is, theexperiment successfully retargeted the epithelial cell A431.

Example F-4 Preparation of Deletion HBsAg L Protein by Transformation inYeast Cells

Genes that express the deletion HBsAg L proteins Δ21-153, Δ33-153, andΔ50-153 (proteins (a) through (c)) which showed desirable levels ofprotein expression in Cos7 cells were obtained by cleaving the plasmidat the XhoI sites. The genes so obtained were inserted at the XhoI sitesof the yeast expressed plasmids pGLDLIIP39-RcT (see FIG. 20), which werethen transferred to S. cerevisiae AH22R⁻ strain by a spheroplast method.The transformants were incubated for 3 days in industrial media High-Piand another 3 days in 8S5N-P400 media. The cultured cells were collectedand disrupted with glass beads. Then, the cell extract was measured toconfirm antigenicity and the level of expression. Antigenicity wasmeasured with a cultured yeast enzyme immunoassay device IMx (DainabotCo. Ltd.), and the level of protein expression was measured by SDS-PAGEand Western blotting (using anti-S protein antibody as the primaryantibody, and AP tagged anti-mouse IgG antibody as the secondaryantibody) (see FIG. 21 and FIG. 22).

Additionally, two kinds of plasmids were constructed using the NotIsites: a plasmid for displaying a ZZ domain gene of protein A; and aplasmid for displaying EGF (see FIG. 20). In sum, the following plasmidswere constructed (expression plasmid for efficiently expressing deletionHBsAg L protein in yeasts): pGLDLIIP39-RcT-Δ50-153;pGLDLIIP39-RcT-Δ33-153, pGLDLIIP39-RcT-Δ21-153;pGLDLIIP39-RcT-Δ50-153-ZZ; pGLDLIIP39-RcT-Δ33-153-ZZ;pGLDLIIP39-RcT-Δ21-153-ZZ; pGLDLIIP39-RcT-Δ50-153-EGF;pGLDLIIP39-RcT-Δ33-153-EGF; and pGLDLIIP39-RcT-Δ21-153-EGF. In addition,pGLDLIIP39-RcT-Δ3-66 was constructed as a control. These yeast-expressedplasmids were transferred into S. cerevisiae AH22R⁻ strain by aspheroplast method. The transformants were incubated for 3 days inindustrial media High-Pi and another 3 days in 8S5N-P400 media. Thecultured cells were collected and disrupted with glass beads, and thecell extract was measured to confirm the level of protein expression bymeasuring antigenicity with a cultured yeast enzyme immunoassay deviceIMx (Dainabot Co. Ltd.) (see FIG. 21 and FIG. 22).

The enzyme immunoassay confirmed formation of deletion particles. Thelevels of antigenicity for the deletion HBsAg L proteins (deletion HBsAgparticles) Δ21-153, Δ33-153, and Δ50-153 compared to that of wild-typeparticles (LAg in the drawings) (see FIG. 21 and FIG. 22). The deletionHBsAg particles A3-66 used as a control produced no transformant (notshown). The deletion HBsAg particles Δ50-153 are particularlyadvantageous since its level of protein expression, combined with aconsiderably large amount of deletion HBsAg particles displaying a ZZdomain (Δ50-153+ZZ), exceeds that of the wild-type.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a drug whose particlesurface displays an antibody such as a cancer specific antibody. Thedrug can be used by a convenient method of intravenous injection tospecifically and effectively treat specific diseased cells or tissues.The invention is a great leap forward from conventional gene therapy inthat it does not require any surgical operation, and that the risk ofside effect is greatly reduced. The drug is therefore usable in clinicalapplications in its present form.

1. A hollow nanoparticle for delivering a substance to a cell,comprising a modified HBV surface antigen large (HBsAg L) protein formedinto a particle, an antibody displayed on the surface of the particle,and a substance encapsulated inside the particle, wherein the modifiedHBsAg L protein includes a replacement of amino acids 50 to 153 in apre-S region of the HBsAg L protein, corresponding to HBV serotype y, bya ZZ tag consisting of SEQ ID NO: 29, and wherein the antibody binds tothe ZZ tag fused to the HBsAg L protein.
 2. The hollow nanoparticle ofclaim 1, wherein the antibody is a single chain antibody.
 3. The hollownanoparticle of claim 1, wherein the modified HBsAg L protein isexpressed in a eukaryotic cell.
 4. The hollow nanoparticle of claim 3,wherein the eukaryotic cell is selected from a group consisting of ayeast cell, an insect cell, and an animal cell.
 5. The hollownanoparticle of claim 1, wherein the substance comprises a gene.
 6. Thehollow nanoparticle of claim 5, wherein the gene comprises a gene ofthymidine kinase derived from simplex herpes virus.
 7. A hollownanoparticle comprising a modified HBV surface antigen large (HBsAg L)protein formed into a particle, wherein the modified HBsAg L proteinincludes a replacement of amino acids 50 to 153 in a pre-S region of theHBsAg L protein, corresponding to HBV serotype y, by a ZZ tag consistingof SEQ ID NO: 29.